CN105136710A - Determination method of hydroxypropyl chitosan substitution degree - Google Patents
Determination method of hydroxypropyl chitosan substitution degree Download PDFInfo
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- CN105136710A CN105136710A CN201510575791.5A CN201510575791A CN105136710A CN 105136710 A CN105136710 A CN 105136710A CN 201510575791 A CN201510575791 A CN 201510575791A CN 105136710 A CN105136710 A CN 105136710A
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- 238000001514 detection method Methods 0.000 claims abstract description 13
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- 238000001228 spectrum Methods 0.000 claims description 19
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- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The invention discloses a determination method of hydroxypropyl chitosan substitution degree. The determination method of the hydroxypropyl chitosan substitution degree comprises the following steps: (1) drying a hydroxypropyl chitosan sample; (2) establishing a detection model: a. obtaining an infrared absorption spectrogram of the sample by using an infrared absorption spectrometer; b. measuring a peak-intensity absorbance value A1 of methyl characteristic peaks 2971 to 2962cm<-1> by using a valley connecting line of an entire waveform 3880 to 1880cm<-1> as a baseline; measuring an absorbance value A2 of the entire waveform 1740 to 1515cm<-1> including amide and amino characteristic peaks by using the valley connecting line as the baseline; obtaining a logarithm X of 100 times of the ratio of A1 to A2; c. measuring the substitution degree Y of the hydroxypropyl chitosan sample through a standard method; d. establishing a linear regression model Y=kX+b by Y to X to obtain specific numerical values of k and b in the model; (3) substituting the ratio X obtained through the infrared absorption spectrogram of the dried to-be-tested sample into the linear regression model to obtain the substitution degree Y. The method is simple, convenient, quick and accurate, the sample does not need to be precisely weighed and dissolved, and the method can also be applied to handheld infrared absorption spectrometers.
Description
Technical field
The invention belongs to the detection method Study and appliance field of hydroxypropyl chitosan degree of substitution, relate to a kind of assay method of hydroxypropyl chitosan degree of substitution, be specifically related to a kind of method of infrared absorption spectrometry hydroxypropyl chitosan degree of substitution.
Background technology
Shitosan has good biocompatibility, biodegradability, stability, security, but it is water-soluble poor, can only acid medium be dissolved in, and shitosan hydroxypropylation be generated hydroxypropyl chitosan, then its dissolubility be can improve, thus its application and development process greatly widened.Show after deliberation, hydroxypropyl chitosan has dissolubility, hydroscopicity, moisture retention, film forming, foamability, scavenging free radicals, antibiotic property, adsorbability (ChengZ, etal.Effectsofhydroxypropyldegreeonphysiochemicalactivit iesofchitosanfromsquidpens.InternationalJournalofBiologi calMacromolecules, 2014,65:246 – 251; DongY, etal.Influenceofdegreeofmolaretherificationoncriticalliq uidcrystalbehaviorofhydroxypropylchitosan.EuropeanPolyme rJournal, 2001,37 (8): 1713 – 1720), all have wide application prospects in fields such as medicine, cosmetics, food, papermaking, weavings (Song Fu comes. the physicochemical property of shitosan and type hydrogel, hemostatic function and Study on biocompatibility. and functional material, 2014,45 (9): 09065-09069; Glad refreshing etc. the impact of the preparation of hydroxypropyl chitosan, sign and corneal Growth of Cells thereof. Chinese Marine University's journal, 2007,37:131-134; Tang Xinfeng. hydroxypropyl chitosan preparation and the application as Neutral Papermaking auxiliary agent thereof. Wuhan University's Master's thesis, 2005; The fabrication & properties of season jasmine .HPCTS and the applied research in textile finishing thereof. Sichuan University's Master's thesis, 2005; Yuan Yihua etc. the preparation of hydroxypropyl chitosan and moisture absorption thereof, moisture retention is studied. meticulous and specialty chemicals, 2005; ZhangL, etal.Effectoforallyadministeredhydroxypropylchitosanonth elevelsofiron, copper, zincandcalciuminmice.InternationalJournalofBiologicalMac romolecules, Volume64, March2014, Pages25 – 29).
When discovery hydroxypropyl chitosan has so potential using value, related researcher will inevitably study the impact of the degree of substitution of hydroxypropyl on the shitosan quantity of each Glucosamine and the hydroxypropyl that 2-Acetamido-2-deoxy-D-glucose molecule is connected (in the hydroxypropyl glycan molecule) on hydroxypropyl chitosan performance and application thereof further, but, the research report of rarely seen this respect.Even if there is bibliographical information, as being published in " the hydroxypropyl chitosan biodegradation Journal of Sex Research of different degree of substitution " (2004 on " Chinese Sea medicine ", 1:33-36) just have employed the assay method of the hydroxypropyl of " Chinese Pharmacopoeia " two, namely, by oxidization combination alkali titration, but and have no degree of substitution test result.Foreign literature Effectsofhydroxypropyldegreeonphysiochemicalactivitiesof chitosanfromsquidpens.InternationalJournalofBiologicalMa cromolecules, 2014,65:246 – 251; DongY, etal.Influenceofdegreeofmolaretherificationoncriticalliq uidcrystalbehaviorofhydroxypropylchitosan.EuropeanPolyme rJournal, 2001,37 (8): 1713 – 1720) when studying degree of substitution problem, mainly based on elemental microanalysis method.
The assay method of the hydroxypropyl chitosan degree of substitution of bibliographical information is had to have elemental microanalysis method (Zhang Yongqin at present, Zhang Kun. the degree of substitution of elemental microanalysis method Simultaneously test hydroxypropyl chitosan and moisture. assay office, 2014,33 (8): 978-980), nucleus magnetic hydrogen spectrum method (DongY, etal.Influenceofdegreeofmolaretherificationoncriticalliq uidcrystalbehaviorofhydroxypropylchitosan.EuropeanPolyme rJournal, 2001,37 (8): 1713 – 1720; PengY, etal.Preparationandantimicrobialactivityofhydroxypropylc hitosan.CarbohydrateResearch, 2005,340 (11): 1846 – 1851) and published absorption photometry (number of patent application: 201410841212.2).
Hydroxypropyl chitosan is generally high molecular polymer, interior, the intermolecular different Interaction of substituents of molecule is complicated, therefore, in the analytical approach of proton nmr spectra, be easy to introduce error by the Hydrogen Proton quantity that the relative absorbance intensity of different chemical displacement place Hydrogen Proton quantitatively calculates under corresponding environment, and significantly affects the accurate calculating of degree of substitution.And elemental microanalysis method only uses the C in sample, N mass ratio, and the measured value of H% and calculated value are contrasted, parameter is single and by the impact of sample degree of substitution height and moisture, therefore, reference and the comparative studies of this other assay method of degree of substitution is usually used in.Above-mentioned two kinds of method testing costs are all higher, are not suitable as the conventional sense in enormous quantities of hydroxypropyl chitosan manufacturing enterprise and research unit.
The testing cost of infrared absorption spectroscopy is relatively low and precision need not take, dissolve shitosan sample, simple and efficient to handle, particularly also be applicable to hand-held infrared absorption spectrometer, therefore, this method is significant to the quality control of the manufacturing enterprise of hydroxypropyl chitosan and application research and development, the quick detection of quality testing department, the scientific research of scientific research institutions.
But namely start the research to hydroxypropyl chitosan from the eighties both at home and abroad, researcher generally applies the method one of of its infrared absorption spectra as structural characterization, but has no the relevant report utilizing this method to measure hydroxypropyl chitosan degree of substitution up to now.Trace it to its cause, denier impurity on the one hand in hydroxypropyl chitosan sample can make its infrared spectrogram " penetrate deeply into all things " and display, and the peak shape at direct effect characteristics peak and peak position change (Zhang Yongqin, Zhang Kun. the impact that isolation and purification method measures hydroxypropyl chitosan structure and degree of substitution. Qingdao University of Science and Technology's journal, 2015,36 (3): 251-254); On the other hand, hydroxypropylation itself also can make it because of overlapping, the distortion mutually of each characteristic peak, migration etc., and is difficult to find certain constant architectural feature peak as the calculating of reference peak for degree of substitution.Therefore, determining that methyl characteristic peak is as under the prerequisite analyzing peak, finds suitable reference peak and determines that the integration method of the two is also key of the present invention.
Summary of the invention
In order to solve above-mentioned the deficiencies in the prior art, primary and foremost purpose of the present invention be to provide a kind of easy fast, accurately, the detection method of the hydroxypropyl chitosan degree of substitution of low cost.
Object of the present invention is achieved through the following technical solutions:
An assay method for hydroxypropyl chitosan degree of substitution, is characterized in that, comprises the following steps:
(1) dry hydroxypropyl chitosan sample is to remove moisture, wherein each sample is all reacted by hydroxypropylation and takes off alkali purification and obtains, and derives from the known same raw materials of chitosan of deacetylation and/or deacetylation differs with this raw materials of chitosan the shitosan being no more than 3%;
(2) set up detection model
A. the infrared absorpting light spectra with the hydroxypropyl chitosan sample of different degree of substitution dried in utilizing infrared absorption spectrometer acquisition step (1);
B. utilize the application software of infrared absorption spectrometer, from a step, the infrared absorpting light spectra that obtains records with whole waveform 3880-1880cm
-1trough line be the methyl characteristic peak 2971-2962cm of baseline BL1
-1the absorbance A1 of peak intensity; Record the whole waveform 1740-1515cm comprising acid amides characteristic peak and amino characteristic peak
-1the absorbance A2 of area, with its trough line for baseline BL2; Thus obtain the logarithm X of 100 times of the ratio of A1 and A2;
C. the degree of substitution Y of hydroxypropyl chitosan sample is recorded with standard method;
D. with the ratio X obtained in b step, linearly good regression model is set up to the degree of substitution Y of the hydroxypropyl chitosan sample obtained in step c, i.e. Y=kX+b, thus obtain the concrete numerical value of k and b in model;
(3) detect: hydroxypropyl chitosan sample to be measured is dry according to method (1), from (2), a step obtains infrared absorpting light spectra, obtaining ratio X according to b step in (2), substituting into the linear regression model (LRM) of Step d (2), namely by calculating the degree of substitution Y of hydroxypropyl chitosan sample to be measured.
Preferably, the method for dry hydroxypropyl chitosan sample is, at 80 DEG C of dry 4-5 hour in baking oven, then rapid temperature increases to 105 DEG C dry again 1.5-2 hour, takes out in the exsiccator that calcium oxide is housed for subsequent use.
Preferably, the infrared absorption spectrometer that (2), a step uses is Fourier transform infrared spectrometer,
Preferably, (2) in the testing conditions of infrared absorption spectrometer that uses in a step be: sweep limit 4000-400cm
-1, scan 16-32 time, resolution is 4cm
-1.
Preferably, before utilizing infrared absorption spectrometer test hydroxypropyl chitosan sample, background detection is carried out.
Preferably, carry out buckleing background process to the collection of illustrative plates in described infrared absorpting light spectra
Preferably, described standard method is elemental microanalysis method.
Compared with prior art, tool has the following advantages in the present invention:
(1) easy and simple to handle, the degree of substitution that detects hydroxypropyl chitosan quickly;
(2) need not carry out the step such as precision weighing, dissolving to sample
(3) be also applicable to the detection of hand-held infrared absorption spectrometer
(4) be applicable to the sample detection of manufacturing enterprise to the quality control of hydroxypropyl sugar product, applied research and development and quality inspection organization.
Accompanying drawing explanation
Fig. 1 is the infrared absorpting light spectra (representing with transmittance) of the hydroxypropyl chitosan of shitosan and different degree of substitution.No. 0 is shitosan sample, No. 1-7 hydroxypropyl chitosan sample increased progressively successively for degree of substitution.
Fig. 2 is the infrared absorpting light spectra (representing with absorbance) of hydroxypropyl chitosan.
Fig. 3 be in Fig. 2 hydroxypropyl chitosan at 4000-1400cm
-1enlarged drawing in scope.Wherein A1 is with whole waveform 3880-1880cm
-1trough line be the absorbance of the peak height of the methyl characteristic peak of baseline BL1, A2 is the whole waveform 1730-1530cm comprising acid amides characteristic peak and amino characteristic peak
-1with the absorbance that its trough line is the area of baseline BL2.
Fig. 4 is the linear regression typical curve of hydroxypropyl chitosan degree of substitution, and wherein, X is that Lg (100 × A1/A2), Y are for utilizing the degree of substitution as the corresponding hydroxypropyl chitosan sample measured by the elemental microanalysis method of standard method.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto embodiment, every change or equivalent alternative all in protection scope of the present invention not deviating from the present invention's design.
Embodiment 1
Reagent: KBr is optical voidness, and antifebrin is standard substance, all derives from traditional Chinese medicines reagent company limited.Shitosan, derives from Laizhou, Shandong sea Lik-Sang Tetramune company limited; Hydroxypropyl chitosan, respectively with No. 0, shitosan for raw material, by reacting different time with epoxypropane under 45-60 DEG C of condition in the basic conditions, the hydroxypropyl chitosan No. 1-No. 7 of the different degree of substitution utilizing de-alkali purification to obtain.
Instrument: the present invention is applicable to various Fourier transform infrared spectrometer, instrument of the present invention is the Fourier transform infrared spectrometer TENSOR27 of German Brooker (Bruker) company; The deacetylation of analyses shitosan adopted in standard method and the degree of substitution of hydroxypropyl chitosan, be complete in the test center (national open laboratory) of approved qualified bioenergy research institute of the Chinese Academy of Sciences of State Metrological Bureau, instrument is the VarioELcube elemental analyser of German Elementar company.
(1) dry shitosan sample, hydroxypropyl chitosan sample are to remove moisture;
Shitosan, the hydroxypropyl chitosan very easily moisture absorption, therefore, should first fully dryly remove its moisture before sample test.Concrete drying means is for getting appropriate hydroxypropyl chitosan sample in centrifuge tube, be placed in baking oven 80 DEG C of dryings 5 hours, then rapid temperature increases to 105 DEG C, 105 DEG C dry 1.5 hours again, rapidly the lid of centrifuge tube is closed after drying, put into the exsiccator that calcium oxide is housed for subsequent use.
(2) set up detection model
A. utilize infrared absorption spectrometer obtain step (1) in dried shitosan and there is the infrared absorpting light spectra of hydroxypropyl chitosan sample (unless refered in particular to below, being all referred to as sample) of different degree of substitution
First, empty sheet is suppressed.Get appropriate KBr in mortar, be ground to and no longer include macroscopic granule, to ensure uniformity coefficient and the transparency of compressing tablet.After having ground, loaded in sample groove by KBr powder, shakeout, put into sheeter central authorities after loading onto other parts, rotating screw bolt, to fix mould, presses hand lever up and down, and make pressure between 12-25MPa, time controling is at 0.5-1min.By the KBr sheet demoulding pressed, the detecting device with tweezers KBr sheet being put into infrared absorption spectrometer carries out background detection.Testing conditions is: sweep limit 4000-400cm
-1, scan 16 times, resolution is 4cm
-1;
Secondly, compacting sample strip.Get appropriate KBr and sample in mortar, detect with above-mentioned same step compressing tablet.Wherein, sample is respectively No. 0, shitosan sample, No. 1-7, the hydroxypropyl chitosan sample that degree of substitution increases progressively successively.Sample introduction is generally 1-2mg, requires that in the infrared absorpting light spectra obtained, most absorption peak is in 30%-100% range of transmittance;
Finally, obtained infrared absorption spectrum is carried out detain the infrared absorpting light spectra that background process obtains required sample.
B. the infrared absorpting light spectra that the application software OPUS process of infrared absorption spectrometer obtains from step a is utilized, as shown in Figure 1.Transfer transmittance modes to absorbance patterns, namely obtain the infrared absorpting light spectra represented with absorbance, as shown in Figure 2.Fig. 2 is got 4000-1400cm
-1region is amplified, and namely obtains Fig. 3.In figure 3, with whole waveform 3880-1880cm
-1trough (3841cm
-1, 1889cm
-1) line is baseline BL1, records methyl characteristic peak 2968-2966cm
-1the absorbance A1 of peak intensity; With whole waveform 1740-1515cm
-1trough (1730cm
-1, 1530cm
-1) line is the absorbance A2 that baseline BL2 records its peak area; Thus obtain the logarithm X of 100 times of the ratio of A1 and A2, wherein the truth of a matter of logarithm is greater than 0 and is not equal to 1, e.g., can get denary logarithm, that is, X=lg (100 × A1/A2), also can get with e is the logarithm at the end, that is, X=Ln (100 × A1/A2), etc.;
C. utilize the elemental microanalysis method in standard method to record the deacetylation of shitosan sample, and then record the degree of substitution Y of hydroxypropyl chitosan sample;
First, production standard curve: using antifebrin as standard substance, sets up mass gradient typical curve; Then, take 2-4mg sample (No. 0-No. 7) respectively and put into Xi Zhouzhong, by tin boat fold seals after correct amount, put into sample cell and measure, each sample does three Duplicate Samples, and end product is averaged.First being 95.1% by calculating the deacetylation of No. 0, shitosan sample, then can calculating the degree of substitution Y of No. 1-7, hydroxypropyl chitosan sample on this basis.
D. with the ratio X obtained in step b, linearly good regression model is set up to the degree of substitution Y of the hydroxypropyl chitosan sample obtained in step c, i.e. Y=kX+b, thus obtain k and b in model.If X is the logarithm that is the truth of a matter with 10, then k=5.2113, b=-0.8335, i.e. Y=5.2113X-0.8335, as shown in Figure 4 and Table 1; X is take e as the logarithm of the truth of a matter, then, k=2.2632, b=-0.8335, as shown in table 2.
Table 1. fourier transform infrared spectroscopy (FTIR) linear regression model (LRM) (X obtained for the truth of a matter with 10)
Table 2. fourier transform infrared spectroscopy (FTIR) linear regression model (LRM) (taking e as the X that the truth of a matter obtains)
(3) detect: hydroxypropyl chitosan sample to be measured is dry according to method (1), infrared absorpting light spectra is obtained by a step in (2), and obtain ratio X according to b step in (2), substitute into the linear regression model (LRM) Y=5.2113X-0.8335 that (2), Step d obtains, namely by calculating the degree of substitution of hydroxypropyl chitosan sample to be measured.
Embodiment 2
Hydroxypropyl chitosan sample A, B, C, D, E, F to be measured, all obtained by hydroxypropylation and de-alkali purification.The deacetylation of its raw materials of chitosan is respectively 92.2%, 93.8%, 94.4%, 95.1%, 96.3%, 97.6%.Compared with setting up the raw materials of chitosan (95.1%) of equation of linear regression hydroxypropyl chitosan sample used, its deacetylation differs 3.05% at most.By according to the dried hydroxypropyl chitosan to be measured of method (1) by Fourier transform infrared spectrometer according to (2) a step and by background detection and button background process, thus obtain infrared absorpting light spectra.According to the method for (2) b step, in OPUS software, with 3841-1889cm
-1methyl characteristic peak 2971-2962cm is obtained for baseline BL1 chooses R baseline integral method
-1the absorbance A1 of peak intensity, choose the absorbance A2 that C baseline integral method records the integral area of acid amides characteristic peak, thus obtain the logarithm value X of 100 times of the ratio of A1 and A2, utilize equation of linear regression Y=5.2113X-0.8335, thus calculate the degree of substitution of hydroxypropyl chitosan sample to be measured, as shown in table 3.
The degree of substitution of table 3 hydroxypropyl chitosan sample to be measured
Embodiment 3
The dealkalize method purification process of hydroxypropyl chitosan
Raw materials of chitosan need carry out de-alkali purification after reacting with epoxypropane generation hydroxypropylation in the basic conditions.The dealkalize method purification process of sample comprises in alcohol wash ketone heavy dialysis dealkalize method and part and ultrafiltration dealkalize method.
In alcohol wash ketone heavy dialysis dealkalize method, first by product mixture suction filtration with except desolventizing and residual alkali.Crude product after suction filtration is dissolved in water, filters, ethanol is added in filtrate, make it the ethanol containing 75%, add acetone while stirring and the swelling shitosan of generation is precipitated out, filter, precipitation is joined again in 75% ethanol, so repeatedly, until the ethanol water of hydroxypropyl chitosan is aobvious neutral, then with acetone precipitation out, to precipitate soluble in water, obtain hydroxypropyl chitosan sample through freeze drying.
In part and in dialysis dealkalize method, first by product mixture suction filtration with except desolventizing and residual alkali.Crude product after suction filtration is dissolved in water, filters, crude product after suction filtration is dissolved in water, filters, in filtrate, add hydrochloric acid makes filtrate be partially neutralized to more than pH10, then, by ultrafiltration dealkalize, slough NaCl simultaneously, by filtrate reduced in volume, namely obtain hydroxypropyl chitosan sample through freeze drying or spraying dry.
Embodiment 4
For the dealkalize method purification process of the hydroxypropyl chitosan sample containing residual acid
Obtaining the situation of the hydroxypropyl chitosan containing residual acid for not purifying by the method for embodiment 3, can aqueous slkali be added, due in and terminal be difficult to judge, therefore need excessive interpolation aqueous slkali, and then carry out dealkalize according to the method for embodiment 3.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; other is any do not deviate from Spirit Essence of the present invention and principle under do change, modification, substitute, combine, simplify the substitute mode that all should be equivalence, be included within protection scope of the present invention.
Claims (7)
1. an assay method for hydroxypropyl chitosan degree of substitution, comprises the following steps:
(1) dry hydroxypropyl chitosan sample is to remove moisture, wherein each hydroxypropyl chitosan sample is all reacted by hydroxypropylation and takes off alkali purification and obtains, and derives from the known same raw materials of chitosan of deacetylation and/or deacetylation differs with its raw materials of chitosan the shitosan being no more than 3%;
(2) set up detection model
A. the infrared absorpting light spectra with the hydroxypropyl chitosan sample of different degree of substitution dried in utilizing infrared absorption spectrometer acquisition step (1);
B. utilize the application software of infrared absorption spectrometer, from a step, the infrared absorpting light spectra that obtains records with whole waveform 3880-1880cm
-1trough line be the methyl characteristic peak 2971-2962cm of baseline
-1the absorbance A1 of peak intensity; Record the whole waveform 1740-1515cm comprising acid amides characteristic peak and amino characteristic peak
-1the absorbance A2 of area, with its trough line for baseline; Thus obtain the logarithm X of 100 times of the ratio of A1 and A2;
C. the degree of substitution Y of the hydroxypropyl chitosan sample in a step is recorded with standard method;
D. with the ratio X obtained in b step, linearly good regression model is set up to the degree of substitution Y of the hydroxypropyl chitosan sample obtained in step c, i.e. Y=kX+b, thus obtain the concrete numerical value of k and b in model;
(3) detect: hydroxypropyl chitosan sample to be measured is dry according to method (1), from (2), a step obtains infrared absorpting light spectra, ratio X is obtained according to b step in (2), substitute into the linear regression model (LRM) that (2), Step d obtains, namely by calculating the degree of substitution Y of hydroxypropyl chitosan sample to be measured.
2. method according to claim 1, it is characterized in that, the method of described dry hydroxypropyl chitosan sample is at 80 DEG C of dry 4-5 hour, and then rapid temperature increases to 105 DEG C dry again 1.5-2 hour, takes out in the exsiccator that calcium oxide is housed for subsequent use by this sample.
3. method according to claim 1, is characterized in that, described infrared absorption spectrometer is Fourier transform infrared spectrometer.
4. method according to claim 1, is characterized in that, the testing conditions of described infrared absorption spectrometer is: sweep limit 4000-400cm
-1, scan 16-32 time, resolution is 4cm
-1.
5. method according to claim 1, is characterized in that, carries out background detection to the infrared absorpting light spectra of obtained hydroxypropyl chitosan sample.
6. the method according to claim 1 or 3 or 4 or 5, is characterized in that, also carries out buckleing background process to the infrared absorpting light spectra of described hydroxypropyl chitosan sample.
7. method according to claim 1, is characterized in that, described standard method is elemental microanalysis method.
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